Neurotoxic effects of TDP-43 overexpression in C-elegans
Neurotoxic effects of TDP-43 overexpression in C-elegans
复制标题
DOI：
10.1093/hmg/ddq230
复制
发表时间：
2010-08-15
影响因子：
3.5
通讯作者：
Link, Christopher D.
中科院分区：
文献类型：
--
作者：
Ash, Peter E. A.;Zhang, Yong-Jie;Link, Christopher D.
RNA结合蛋白TDP-43与多种神经退行性疾病相关，包括肌萎缩侧索硬化和额颞叶痴呆。我们已经在秀丽隐杆线虫中设计了人TDP-43蛋白的泛神经元表达，目的是产生TDP-43功能和神经毒性的方便的体内模型。具有人TDP-43的神经元表达的转基因蠕虫表现出“不协调”表型，并且具有异常的运动神经元突触。秀丽隐杆线虫含有TDP-43的一个推定的直系同源物，命名为TDP-1，我们表明它可以支持CFTR在基于细胞的测定中的选择性剪接。TDP-1的神经元过表达也导致不协调的表型，而TDP-1基因的遗传缺失不影响运动或改变运动神经元突触。通过使用不协调的表型作为TDP-43过表达神经毒性的读数，我们研究了特定的TDP-43结构域和亚细胞定位对毒性的贡献。全长（野生型）人TDP-43在C. elegans被定位于细胞核。RNA识别结构域（RRM 1或RRM 2）的缺失完全阻断神经毒性，C-末端区域的缺失也是如此。这些缺失的TDP-43变体仍然在细胞核中积累，尽管它们的亚核分布发生了改变。有趣的是，TDP-1C-末端序列与缺失其C-末端结构域的TDP-43的融合恢复了C. elegans和CFTR剪接。野生型全长TDP-43在哺乳动物细胞（分化的M17细胞）中的过表达也可导致细胞毒性。我们的研究结果表明，在体内TDP-43的神经毒性，可导致核活性的过表达全长蛋白质。
RNA-binding protein TDP-43 has been associated with multiple neurodegenerative diseases, including amyotrophic lateral sclerosis and frontotemporal lobar dementia. We have engineered pan-neuronal expression of human TDP-43 protein in Caenorhabditis elegans, with the goal of generating a convenient in vivo model of TDP-43 function and neurotoxicity. Transgenic worms with the neuronal expression of human TDP-43 exhibit an 'uncoordinated' phenotype and have abnormal motorneuron synapses. Caenorhabditis elegans contains a single putative ortholog of TDP-43, designated TDP-1, which we show can support alternative splicing of CFTR in a cell-based assay. Neuronal overexpression of TDP-1 also results in an uncoordinated phenotype, while genetic deletion of the tdp-1 gene does not affect movement or alter motorneuron synapses. By using the uncoordinated phenotype as a read-out of TDP-43 overexpression neurotoxicty, we have investigated the contribution of specific TDP-43 domains and subcellular localization to toxicity. Full-length (wild-type) human TDP-43 expressed in C. elegans is localized to the nucleus. Deletion of either RNA recognition domain (RRM1 or RRM2) completely blocks neurotoxicity, as does deletion of the C-terminal region. These deleted TDP-43 variants still accumulate in the nucleus, although their subnuclear distribution is altered. Interestingly, fusion of TDP-1 C-terminal sequences to TDP-43 missing its C-terminal domain restores normal subnuclear localization and toxicity in C. elegans and CFTR splicing in cell-based assays. Overexpression of wild-type, full-length TDP-43 in mammalian cells (differentiated M17 cells) can also result in cell toxicity. Our results demonstrate that in vivo TDP-43 neurotoxicity can result from nuclear activity of overexpressed full-length protein.